Systems and methods for sidesstream dark field imaging

ABSTRACT

The present application discloses systems and methods for the comprehensive monitoring of the microcirculation in order to assess the ultimate efficacy of the cardiovascular system in delivering adequate amounts of oxygen to the organ cells. In some cases, system embodiments may utilize reflectance avoidance by reflectance filtering, such as OPS imaging or Mainstream Dark Field imaging, or by Sidestream Dark Field imaging, which utilizes external direct light on the tip of the light guide to achieve reflectance avoidance whereby incident and reflected light do not travel down the same pathway.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/273,118, filed on Oct. 13, 2011, and naming Can Ince asinventor, which is a continuation of U.S. patent application Ser. No.10/956,610, now U.S. Pat. No. 8,064,976, filed on Oct. 1, 2004, andnaming Can Ince as inventor, which claims priority to U.S. ProvisionalPatent Application Ser. No. 60/508,347, filed on Oct. 3, 2003, andnaming Can Ince as inventor, and which also claims priority to U.S.Provisional Patent Application Ser. No. 60/557,792, filed on Mar. 29,2004, and naming Can Ince as inventor, all of which are incorporated byreference herein in their entirety.

BACKGROUND

Currently, physicians typically monitor a number of systemic (e.g. themacrocirculation) hemodynamic parameters when diagnosing and monitoringof the hemodynamic condition of patients. For example, blood flow andpressure are regularly monitored. In addition, a blood sample may bewithdrawn from the patient to determine the oxygenation of the red bloodcells as well as the oxygen carrying capacity of the circulating blood.Furthermore, a biopsy may be required to determine the functional stateof tissue cells (e.g. the oxygenation and viability of tissue cells) ofthe organ system.

While monitoring these macrohemodynamic parameters has proven successfulin diagnosing and monitoring a number of conditions, severalshortcomings have been identified. For example, examiningmacrocirculatory parameters provides little or no information relativeto the microcirculatory (i.e. hemodynamics and structure of bloodvessels smaller than 250 microns) characteristics of patients. Currentresearch has shown that distress at the microcirculatory level involvedin a large number of disease states is not discoverable by monitoringmacrocirculation. As such, diseases or other complications evidentthrough microcirculatory monitoring may go undetected and untreated.

It is believed, for example, that improved clinical observation of themicrocirculation of human organs would be extremely useful in assessingstates of shock such as septic, hypovolemic, cardiogenic and obstructiveshock in patients and in guiding resuscitation therapies aimed atcorrecting this condition. In particular, it has been found that theactive recruitment of the microcirculation maybe an important componentof resuscitation. Additionally, improved clinical observation of themicrocirculation would be helpful in observing gross circulatoryabnormalities in pathologies such as tumors and cardiovascular disease.

To fully monitor the function of the microcirculation, that is thestructure and perfusion of vessels smaller than 250 micrometers, inaddition to measuring blood flow it is important to measure and asseswhether the blood cells are successful in transporting their oxygen tothe microcirculation and thereafter to the surrounding tissue cells. Ofparticular importance is the assessment of the perfusion of thecapillaries, which are between approximately 5 to 10 micrometers,because it is at this level that oxygen is transported by the red bloodcells to the tissue cells of the organ for the purposes of respirationand survival. Monitoring the functional state of the microcirculationcan thus be regarded as monitoring the ultimate efficacy and function ofthe cardiovascular system to deliver adequate amounts of oxygen to theorgan cells.

It is believed, for example, that improved and comprehensive imaging ofthe properties of the microcirculation would be helpful in observing andassessing the beneficial effects of therapy during the resuscitation ofshock patients. An accurate assessment of both blood flow and oxygenavailability at the level of the microcirculation could thus provide aclinical tool with which to guide resuscitation. A comprehensive way tomonitor the microcirculation could generally provide an improvedclinical diagnostic tool for evaluating and monitoring the functionalstate of the microcirculation in the peri-operative phase of treatment.

To date, there have been limits to a comprehensive monitoring of themicrocirculation in order to provide the benefits discussed above.Specifically, several factors have limited the ability to evaluate theoxygen transport variables of the microcirculation comprehensively. Forexample, devices which contact the surface of the microcirculationinhibit their ability to obtain quantitative information about bloodflow in the various categories of micro-vessels in the microcirculationby impeding flow due to exerted pressure. Furthermore, current devicesand techniques for imaging the microcirculation do not provide theadditional needed information about the oxygen availability in themicrocirculation or about the adequacy of oxygenation of the tissuecells. This information would be very helpful in assessing thefunctional state of the microcirculation, specifically its function inallowing adequate transport of oxygen to the tissue cells. Thus, thereis a need for an improved system and method for a more effective and amore comprehensive clinical observation of the microcirculation whichincludes these parameters.

SUMMARY

The system and method disclosed herein provides comprehensiveinformation about the microcirculation by providing multiple modes ofoptical spectroscopy and imaging in a manner which does not influencethe microcirculation. In one aspect, the system avoids reflection oflight from the tissue in the various imaging modes. This reflectanceavoidance can be provided by reflectance filtering, such as orthogonalpolarization or cross-polarization of light or dark field imaging, or bysidestream dark field imaging, wherein, for example, incident andreflected light may not travel down the same pathway.

In order to image flowing cells in the microcirculation, light has to beilluminated on to the surface of the organs, which is the substrate, anda magnifying lens may be used. Use of a specific wavelength of light(e.g. green light) may allow for better observation of the contrastingred blood cells due to the absorption characteristics of the hemoglobin(hereinafter Hb) in the red blood cells. However, surface reflectionsfrom the substrate can interfere with the ability to clearly visualizethe underlying microcirculation structures and the flowing blood cellstherein. Filtering out of these surface reflection by various methodsallows visualization of the blood flow in the underlyingmicrocirculation on organ surfaces by measurement of the images of themoving cells. Reflectance filtering can be achieved by a number oftechniques which are known to those of skill in the art. The system andmethod disclosed herein may utilize some of these known techniques, butsome novel ones are disclosed as well.

In some embodiments, the system and method utilizes reflectanceavoidance by known techniques of reflectance filtering, such as: 1) OPSimaging, whereby illuminating light and reflected light travel down thesame light guide; or 2) Mainstream Dark Field imaging, wherebyilluminating light and reflected travel down the same light guide butperipheral illumination is achieved by directing the light through, forexample, a hole in a 45° mirror or design of a lens in the illuminatingpathway, which impedes transmission of the light through the middle,and/or a lens which poorly allows transmission of the light through thecentre is put in the pathway of the light to achieve the same effect.

In other embodiments, a novel method of reflectance avoidance isdisclosed which is an alternative to reflectance filtering. This novelapproach, referred to herein as Sidestream Dark Field imaging(hereinafter SDF), utilizes external direct light on the tip of thelight guide to achieve reflectance avoidance whereby incident andreflected light do not travel down the same pathway. This form ofimaging can be provided in combination with a hand-held microscope. Afeature of SDF imaging is that illuminated light and reflected lighttravel via independent pathways. With this modality, the illuminationcan be placed directly on the tissue and the observations can be madeadjacent to it without light crossing over between two paths. Theilluminating light source is typically placed on or near contact withthe tissue. The scattering of the reflected light is thus outside of theimage as most light cross over is below the tissue surface. To date,Mainstream Dark Field imaging has been described as a way of improvingcontrast and lowering surface reflectance, but it typically utilizesillumination and reflectance light paths that travel up and back thesame pathway. In the past, SDF illumination has been applied by ringillumination to improve epi-illumination. It is believed, however, thatit has not been applied to achieve true dark field illumination byilluminating one segment of a substrate and observing in another segmentimages of the microcirculation and its flowing cells. It is believedthat SDF imaging has characteristics which make it superior to othermodes of imaging.

The foregoing reflectance avoidance imaging systems, whether theyutilize OPS, Mainstream Dark Field illumination, or SDF illumination,can be used to enable the comprehensive evaluation of the functionalstate of the microcirculation. This is achieved by an analysis of themoving cells in the images, which permits the quantitative measurementof red blood cell flow in the capillaries, as well as in the largervessels of the microcirculation. This measurement is believed torepresent a truly sensitive measurement which is indicative ofcardiovascular disease and dysfunction. Laser Doppler measurements, forexample, provide an over all flux of moving particles in an unidentifiedcompartment of the circulation, but do not have the specificity formeasurement of cellular perfusion of these smallest capillaries.

The system and method disclosed herein, in providing reflectanceavoidance in combination with optical magnification, provides a superiormethod of measurement of the functional state (e.g.perfusion/oxygenation) of the microcirculation. Next to the measurementof perfusion, morphological characteristics of the microcirculation,such as functional capillary density and micro-vessel morphology, can bemeasured using reflectance avoidance imaging. Homogeneous perfusion ofthe capillaries is a prerequisite for normal function of themicrocirculation and abnormal perfusion or diminished capillaryperfusion is considered an early and sensitive indicator ofcardiovascular disease and failure.

The present application thus relates to a variety of imaging systems foranalyzing the reflectance of an examination substrate. While the imagingsystem disclosed herein may be used to analyze the reflectancecharacteristics of a variety of substrates, it is particularly wellsuited for non-invasively imaging the micro-circulation with a tissuesample.

In one embodiment, the present application discloses a system forimaging the reflectance of a substrate and includes a light source, alight transport body configured to project light from the light sourceto an examination substrate and transmit light reflected and scatteredby the examination substrate, an analysis section in opticalcommunication with the light transport body and having an orthogonalpolarization spectral imaging module or any other of the reflectanceavoidance imaging systems, and at least one of a reflectancespectrophotometry module and a fluorescence imaging module.

In an alternate embodiment, the present application discloses anorthogonal polarization imaging system and includes a light sourceconfigured to emit white light, a first polarizer to polarize the whitelight, a light transport body to transport the polarized light to anexamination substrate and reflect light from an examination substrate, asecond polarizer to filter the light reflected and scattered by theexamination substrate, a filter bank containing at least one wavelengthfilter to filter the reflected light, and an image capture device inoptical communication with the light transport body and configured toimage the reflected light.

In still yet another embodiment, the present application discloses amethod of imaging the reflectance of a substrate and includesilluminating an examination substrate with light, transmitting a portionof light reflected by the examination substrate to a reflectancespectrophotometer, determining a concentration of hemoglobin within theexamination substrate based on a spectral characteristic of theexamination substrate with the reflectance spectrophotometer,transmitting a portion of the light reflected by the examinationsubstrate to an orthogonal polarization spectral imaging module, andmeasuring a flow through a vessel within the examination substrate withan orthogonal polarization spectral imaging module.

In one embodiment, the present application discloses a novel manner ofapplying dark field imaging on the tip of a light guide to provide clearimages of the microcirculation on human organ surfaces. This can beaccomplished by putting light emitting diodes (LED's) around the tip ofthe light guide in combination with a separator so that the illuminatinglight does not enter the reflection light guide directly by surfacereflection, but via the internal structures inside the substrate. Thismodality of reflectance avoidance is a form of dark field imaging whichwe have called Sidestream Dark Field or SDF imaging and providesremarkably clear images of the microcirculation.

In some embodiments, reflectance avoidance imaging is used to obtain amicrocirculatory perfusion index as well as a heterogeneity of flowindex in a device that does not impact flow patterns. This may beaccomplished by using non-contact modes such as, for example, using along focal length, immobilizing the device and substrate by suction atthe tip, or utilizing a spacer between the tissue and the light emittingtip.

In one such embodiment, a novel, “castle” type of spacer is utilized toprovide distance from the examining substrate and to avoid pressure ofthe tip on the substrate. In another embodiment, a needle camera isutilized with a spacer to provide a dark field illumination device. Inyet another embodiment, a suction device is used with reflectanceavoidance imaging techniques.

In another embodiment, a distance spacer is used to achieve reliablecapillary perfusion measurements whereby the tip of the image guide doesnot impede flow in the microcirculation by pressure. In yet anotherembodiment, reflectance avoidance imaging is used in combination with aspace through which fluid, drugs or gasses can be perfused.

In one embodiment, a disposable tip attaches to the end of the deviceand is removed by a release mechanism so that it can be disposed ofwithout having to touch the disposable.

The utilization of reflectance avoidance in the present inventionprovides an improved method of observing microcirculatory hemodynamicsand functional morphology. Image analysis can provide a plurality ofclinical parameters which will have utility for various clinicalconditions. The method and device will assist in providing a perfusionindex such as a measure of functional capillary density, which is thenumber of perfused micro-vessels showing per field observed. Otherparameters include the distribution and heterogeneity of micro-vascularflow, torsion and functional morphology of the blood vessels, thedistribution of diameters of blood vessels, white blood cell kinetics,abnormal red blood cell kinetics (e.g. the presence of micro-vascularcoagulation, sludging or adhesion).

For a comprehensive assessment of the functional state of themicrocirculation, it may be preferable to have more than just perfusioninformation. It would also be useful to have Information about theamount of oxygen bound to the Hb, which can be provided by reflectancespectrophotometry, and information as to whether the tissue cells aregetting sufficient amount of oxygen, which can be provided by measuringtissue CO₂ by sensing the CO₂ in the inside of the disposable, using,for example, CO₂ sensitive fluorescence quenching dyes. The light guidecan then be used to excite the dye with a pulse of light and a detectorwhich measures the CO₂ dependent quenching of fluorescence life timewould provide the measurement. Also, mitochondrial energy states by NADHvia fluorescence imaging can be obtained. Information may be obtainedabout whether there is movement of the red blood cells in themicrocirculation, whether the red blood cells are transporting oxygen(i.e. Hb saturation), and whether the tissue cells are getting enoughoxygen (tissue CO₂ measurement and/or NADH fluorescence imaging).

In some embodiments, reflectance spectrophotometry in conjunction withreflectance avoidance is used to assess the adequacy of oxygenavailability. This may provide for the assessment of microcirculatoryoxygen transport. In some embodiments this can be accomplished by ananalysis of the full reflected spectrum of light (e.g. 400-700 nm). Inother embodiments it is accomplished by an analysis of discretewavelengths outputs of a color sensitive imaging device.Microcirculatory Hb saturation, microcirculatory Hb concentration, andmicrocirculatory hematocrit can all be measured.

In some embodiments, the SDF imaging technique is combined with the useof different wavelengths LED's wherein the images are normalized andBeer Lambert equations are applied.

In some embodiments, NADH fluorescence imaging is used to measure theadequacy of the need for mitochondrial oxygen. This can be used toassess tissue cell dysoxia.

In some embodiments, fluorescence spectroscopy is used for tissue celldiagnostics using endogenous molecules, reporter genes or externalindicator dyes. With appropriate filters, apoptosis can be detected(e.g. via annexin fluorescence), green fluorescent labeled cells used ingene therapy could be located in terms of their efficacy in homing in onthe target.

In one embodiment, a method of imaging the microcirculation by avoidingsurface reflections is combined with reflectance spectrophotometry,Raman spectroscopy, fluorescence spectroscopy and/or other types ofspectroscopic modalities, such as light scatter measurements or opticalcoherence tomography.

In some embodiments, the device is a light guide based system whereinemission and excitation light travels via light guides. In someembodiments, the images are detected at the tip with a tip camera. Thedevice may have a fused silicon lens which will allow 360 nm to pass inorder to enable NADH fluorescence imaging. The device can be either handheld or a flexible endoscopic type.

In addition, to direct contact imaging, the reflectance avoidanceimaging system disclosed herein may also be capable of operating in anon-contact mode which makes use of a spacer to avoid pressure in thetissue surface which may impede blood flow therethrough. Various spaceroptions exist, including;

a. plastic upside down cup attached as disposable;

b. a doughnut shaped spacer (which can be inflatable) with an upsidedown situation/cup;

c. a device (e.g. a plug for around the scope end), such as a concentricring with suction ports, for providing suction through little holesaround the perimeter of the scope thereby immobilizing the perimeter butleaving the microcirculation in the field of view unstressed; or

d. a transparent cushion either solid, air inflatable or filled withfluid.

What is also disclosed is a non-contacting tip for endoscopic use. Inone embodiment, long focus distance imaging can be used to observeretinal microcirculation. This modality can be used to monitor eyediseases and as a monitoring tool during surgery to monitor brainfunction non-invasively. In the retinal application imaging light can bepulsed and small clips of moving images used for monitoring, thusminimizing retinal light exposure.

In one embodiment, the system is configured to operate in a no contactmode without use of a spacer. Thus, the system may be used during brainsurgery or heart surgery. Any movement of the object surface can becorrected by image processing either on-line or after a delay.

In one embodiment the light guide system has an L-shape at the end. Herea 45° mirror creates the bend and LED illumination, using SDF, imagingis present at the tip, with or without a spacer and/or suction module.This embodiment may be used to inspect the sides of hollow spaces suchas is present in the digestive track.

In another embodiment, large objective magnification may be used. Forexample, image processing software may be used to immobilize orstabilize the images, thereby allowing for better image processing ofthe movements.

In still another embodiment, magnification of the substrate image can beinfluenced in several ways. For example, different lenses may be used(different spacer on the tip), or movement of exiting lenses by anopto-mechanical system, or in the electronic mode a larger number ofpixel CCD or CMOS chips, which are known to those of skill in the art,or a larger density of pixels in the chip can be utilized. Movement ofthe CCD or CMOS can also be used to influence magnification.

In still another embodiment, any number of specified color cameras maybe used with the present system. For example, a choice of color orcombination of colors would allow images to be generated of thesaturation of the Hb of the red blood cells in the microcirculation. Afurther embodiment involves looking at only the red output of a colorcamera and to filter out of the rest of the image. This would result inred cells moving in a white background.

Use of a high speed rate (i.e. higher than video rate) can be used forobtaining a proper velocity measurement in conditions in which red bloodcells are moving faster than the video rate.

In some embodiments, a CO₂ measurement of the tissue in the field ofview can be made simultaneously with a reflectance avoidance flowmeasurement and an oxygen availability measurement, such as withspectrophotometry, as a measure of tissue wellness.

In one embodiment, a disposable spacer (e.g. upside down cup) may beemployed. In this embodiment, a CO₂ sensing dye can be impregnated withwhich CO₂ can be sensed within the cup environment. The dye works toprovide a fluorescence decay measurement and the excitation and emissionlight of this dye in the disposable tip can be measured through thelight guide. The CO₂ measurement can be combined with a reflectanceavoidance flow measurement, such as an OPS or SDF imaging basedperfusion measurement. Furthermore, a CO₂ probe may be inserted into thenose of a patient to assess tissue pCO2 and combine this informationwith simultaneously measured perfusion (e.g. by OPS or SDF imaging) andoxygen availability (spectrophotometry) measured sublingually. Inanother embodiment, the CO₂ probe may be used rectally. Thesemeasurements may be made continuously. The sensor may be embedded withina pliable of cushioning material. For example, the sensor may bepositioned within a sponge so as to trap and sense the CO₂ sufficiently.

The CO₂ sensor can be used in the nose and/or rectally as alternativelocations for a separate sensor which is then integrated in themeasurement. This can be in single or in multi mode. The lattertechnique, which makes use of more than one CO₂ sensor, will giveinformation about regional heterogeneity. Using multi locations isbelieved to be a new use of a CO₂ measurement.

In some embodiments, a laser can be included as a therapeutic modality.This can be accomplished, for example, by the use of dark fieldillumination in which the laser goes through the hole in the slantedmirror. In this embodiment, reflectance avoidance imaging is combinedwith the use of the laser for photodynamic therapy (e.g. for cancer) orto coagulate micro-vessels in port wine stains or other cosmeticcorrective procedures.

In another embodiment, reflectance avoidance imaging is used to observethe microstructure of the wound, and temperature is sensed by a solidstate or thermo-sensitive color sensor as well as by opticalspectroscopy to measure the water content. It is thereby that woundperfusion (via e.g. OPS or SDF imaging), wound temperature and edema(water content) will give a comprehensive measurement of the phase ofwound healing and allow assessment of the response to therapy.

In the photodynamic embodiment (where the patient receives aphotosensitive drug) it is possible to apply fluorescence in combinationwith reflectance avoidance for detection of the drug (which accumulatesin tumors) or for enhanced fluorescence in ALA induced protoporphyringfluorescence. Combining a therapeutic laser in the device would make itpossible to deliver photodynamic therapy directly to the area of highfluorescence.

Alternative illumination modalities may include pulsing the LEDillumination in combination with synchronization with a camera for themeasurement of high blood flow velocities. Another alternative includesthe use of an optical foil, acting as a light guide, or other materialwhich may be wrapped around the tip of the probe providing illuminationfrom the side of the tip as an alternative way of illuminating theobject and accomplishing reflectance avoidance. This is similar to themethod which is accomplished by the use of optical fibers placed aroundthe out side of the scope.

Other embodiments which include laser therapies include the use ofreflectance avoidance imaging to verify the effectiveness and allow forthe accurate titration of laser doses. A second example is the use ofphotodynamic therapy for on-line treatment of photosensitized tumors.

In another embodiment, a custom spacer is disclosed in which it ispossible to introduce a drug or gas to the field of observation andmeasure the reactivity of the blood vessels (i.e. losses of which are anindication of poor function). This spacer could be a suction spacerwhich would provide space in the field of view to ensure that there isno contact with the tip and also provide space to inject a drug (formicrocirculatory responsiveness) or for calibration that may be neededfor the embodiment which utilizes a CO₂ sensor placed in the probe.Drugs which can be considered challenges to the microcirculation arevasodilators acting on specific locations of the microcirculation e.g.acetyl choline, lidocaine or nitrate. Others include vasopressors, suchas noradrenaline or dobutamine. This modality can also be used in localtreatment of tumors by application of a topical administration of achemotherapeutic drug.

Measuring the reactivity of the blood circulation to challenges (alsogiven systemically) via, for example, trend measurements, yieldparameters which give additional information than a snap shot analysis.Response to therapy of the microcirculation can be monitoredcontinuously providing on-line information about the functional state ofthe microcirculation during illness.

A further challenge can be induced through a specialized spacer whichapplies a momentary suction pulse and measures the time ofmicrocirculatory refill.

In some embodiments multi-wavelength imaging can be used for themeasurement and analysis of Hb saturation images. The object issequentially or simultaneously illuminated by specific colored LED's,placed in SDF mode, which are chosen at specific wavelengths along theabsorption spectrum of Hb, such that when combined in a composite imagethey provide an image of the distribution of Hb saturation (or Hbconcentration or Hematocrit) of the cells of the microcirculation. Asecond embodiment for achieving the same objective utilizes white light.The reflected light is then split by a multi-wavelength optical memberwhich may consist of mirrors and filters which project two or moreimages each at a different wavelength onto the imaging device to allowreconstituted saturation images to be made.

In one embodiment the use of fluorescence SDF imaging (endogenousleucocyte fluorescence), or observing light scatter, to view differencesbetween cells moving in the circulation (i.e. leucocytes scatter morelight than red blood cells) and combining such imaging, with or withoutfiltering of special wavelengths, optical conditions permit theobservation and quantification of the amount of leucocytes flowing inthe microcirculation. Such a measurement would allow quantification ofthe immune status of the observed field of view by counting the amountof leucocytes and or observing the kinetics of cell sticking or rolling.

In one embodiment, annexin fluorescence can be used for the detection ofapoptotic cells. A combination of fluorescence techniques includes butis not limited to annexin-labeled cells which will allow for thevisualization of apoptotic cells which are directed to programmed celldeath, a precursor to necrosis and cell death. These measurements may beimportant in assessing cell failure in cardiovascular disease, sepsisand in identification and staging of the severity of cancer, or otherstages of diseases such as inflammatory bowel disease. In thisapplication fluorescence labeled annexin is administered to the patient,or applied topically to the site of interest and utilizes thefluorescence mode of the scope. In the fluorescence mode of the scope wedescribe a hand tool (a fluorescence boroscope) such as described forthe reflectance avoidance imaging but in which fluorescence modality isutilized. Reflectance avoidance imaging can be used to improvefluorescence imaging, by filtering or avoiding surface reflections, andcan be applied in the boroscope application or also in fluorescenceendoscopy where, to date, the combination of fluorescence andreflectance avoidance imaging has not been disclosed.

In this embodiment, the appropriate choice of filters can be used toimage mitochondrial energy states (NADH levels) through the use offluorescence. NADH in vivo fluorescence imaging involves dual wavelengthfluorescence combined with reflectance avoidance imaging to correct forchanges in absorption in the image, which can be caused by variation inHb (which is an absorber) in the vessels in the image (results inheterogeneous images). In addition, fluorescence spectrophotometry maybe combined with reflectance avoidance imaging to allow cell diagnosticsduring surgery directly at the bedside. Tissue cell diagnostics willtarget the functional state of the mitochondria by measurement of theenergy of the mitochondria by NADH fluorescence, the gold standard forassessment of tissue dysoxia. Such fluorescence imaging can also be usedin conjunction with diagnostic dyes for identification of apoptosis ortumor cells and reporter genes during gene therapy. Combination offluorescence dyes and cell labeling techniques can be used by thismodality (with appropriate filters) to observe and quantify the degreeof degradation of the glycocalix lining of the endothelia cells. Thisobservation provides a microcirculatory indication of the severity ofcardiovascular disease. Finally measurement of the time course oftransport through the microcirculation of a pulse of fluorescent dyeallows microcirculatory flow at the capillary level to be quantifiedwhen detected by fluorescence.

In some embodiments, reflectance avoidance imaging will be combined withRaman spectroscopy, thereby combining microcirculatory reflectanceavoidance imaging with information about the constituents of thetissues.

The above embodiments can be used in an endoscopy mode. For example,dark field endoscopy, OPS imaging, and\or side illumination can be usedto make observations in the gastric tract, with for example, the L-tipdevice discussed above. Polarization can be achieved at the tip of aflexible endoscope. Dark field illumination can be used in the same wayby concentric illumination. A light conducting foil can be used at theoutside. A 45° mirror can be included at the tip for observation of thesides of the gastric tubes. Thin scopes can be made for pediatrics.

In some embodiments, optical coherence tomography can be used formeasurement of optical path-length using Beer Lambert as a quantitativemeasurement.

Sublingual Near Infra-red Spectroscopy can be used in the transmissionmode or in the reflectance mode to measure total oxygenation of thetongue.

The foregoing methodologies for comprehensive imaging of themicrocirculation provide a useful clinical tool in assessing states ofshock such as septic, hypovolemic, cardiogenic, and obstructive shock inpatients and in guiding resuscitation therapies.

Other objects, features, and advantages of the imaging system and methoddisclosed herein will become apparent from a consideration of thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The imaging system of the present application will be explained in moredetail by way of the accompanying drawings, wherein:

FIG. 1 shows a block diagram of an embodiment of an imaging system foranalyzing light reflected from an examination substrate;

FIG. 2 shows a block diagram of an embodiment of an analyzing section ofan imaging system;

FIG. 3 shows a schematic diagram of an embodiment of a light transportsection configured to project light on and receive reflected light froman examination substrate;

FIG. 4A shows a perspective view of an embodiment of a light transportbody of a light transport section;

FIG. 4B shows a perspective view of an alternate embodiment of a lighttransport body of a light transport section;

FIG. 4C shows a perspective view of another embodiment of a lighttransport body of a light transport section;

FIG. 4D shows a perspective view of still another embodiment of a lighttransport body of a light transport section;

FIG. 5 shows a schematic diagram of another embodiment of a lighttransport section configured to project light on and receive reflectedlight from an examination substrate;

FIG. 6 shows a side view of an alternate embodiment of a light transportbody of a light transport section;

FIG. 7 shows side view of an embodiment of a spacer device coupled to anembodiment of a light transport body;

FIG. 8 shows a side view of another embodiment of a spacer devicecoupled to an embodiment of a light transport body;

FIG. 9 shows a side view of an embodiment of a spacer device configuredto couple to an examination substrate coupled to an embodiment of alight transport body;

FIG. 10 shows a bottom view of an embodiment of the spacer device shownin FIG. 9;

FIG. 11 shows a cross sectional view of an embodiment of an imagingsystem for analyzing reflected light;

FIG. 12 shows a side view of an optical system for use in the an imagingsystem for analyzing reflected light shown in FIG. 10;

FIG. 13 shows a cross sectional view of an embodiment of an imagingsystem for analyzing reflected light having an internal light sourcepositioned therein;

FIG. 14 shows a side cross-sectional view of an embodiment of an imagingsystem configured to permit side stream dark field imaging of an area;

FIG. 15 shows a perspective view of the distal portion of an embodimentof the imaging system shown in FIG. 14;

FIG. 16 shows a cross sectional view of an embodiment of an imagingsystem having one or more illumination sources located withinillumination passages formed in a body;

FIG. 17 shows a cross sectional view of an embodiment of an imagingsystem having a body coupled to handle portion;

FIG. 18 shows a schematic diagram of an embodiment of an imaging systemfor projecting light to a substrate and collecting light therefrom foranalysis;

FIG. 19 shows a perspective view of the distal portion of the imagingsystem shown in FIG. 18;

FIG. 20 shows a perspective view of the distal portion of anotherembodiment of imaging system shown in FIG. 18;

FIG. 21 shows a side cross sectional view of an embodiment of an imagingsystem wherein the distal portion thereof is in contact with anexamination substrate;

FIG. 22 shows a side cross sectional view of an embodiment of an imagingsystem wherein the distal portion includes an engaging device thereon;

FIG. 23 shows a side cross sectional view of an embodiment of an imagingsystem wherein the distal portion is not in contact with the examinationsubstrate;

FIG. 24 shows a block diagram of diagram of an embodiment of an imagingsystem for imaging microcirculation within a structure and analyzinglight reflected from an examination substrate;

FIG. 25 shows a cross sectional view of an embodiment of a cap devicewhich may be affixed to a body of an imaging system;

FIG. 26 shows a perspective view of embodiment of an imaging systemconfigured for sub-surface imaging of an area; and

FIG. 27 shows a side cross sectional view of the imaging system shown inFIG. 26.

DETAILED DESCRIPTION

FIG. 1 shows a block diagram of an embodiment of a reflectance imagingsystem. The imaging system 10 includes an analyzing section 12 and alight transport section 14 configured to project light on and/or receivereflected light from an examination substrate 16. In one embodiment thelight transport section 14 may include an internal light source 18therein configured to provide light of at least one selected wavelengthand/or polarization to the examination substrate 16. Optionally, theinternal light source 18 may be used with or may comprise a source ofwhite or full spectral light thereby enabling spectral analysis of lightreflected by the examination substrate 16. In an alternate embodiment,an external light source 20 may be in optical communication with thelight transport section 14 and configured to illuminate the examinationsubstrate 16. Optionally, the imaging system 10 may include both aninternal light source 18 and an external light source 20. As such, theinternal and external light sources may have the same or differentwavelengths and/or polarizations. In another embodiment, an ancillaryilluminator 22 may be used to illuminate the examination substrate 16.As shown, the ancillary illuminator 22 directly illuminates theexamination substrate thereby foregoing the light transport section 14.The various components of the analyzing section 12 and the lighttransport section 14 will be described in greater detail below.

Referring again to FIG. 1, in one embodiment the analyzing section 12includes any number of modules configured to analyze light reflectedfrom the examination substrate 16 and transported to the analyzingsection 12 by the light transport section 14. In the illustratedembodiment, the analyzing section 12 includes an orthogonal polarizationspectral (OPS) imaging module 30, a reflectance spectrophotometry (RFS)module 32, and a fluorescence (FLS) imaging module 34. Any number ofadditional modules 36 may be included in the analyzing section 12.Exemplary additional modules include, without limitation, Ramanspectroscopy modules, optical coherence tomography modules, dark fieldimaging including side stream dark field imaging (See below), andvarious light scattering measurement modules.

As shown in FIGS. 1 and 2, the OPS imaging module 30 receives a lightsample 40 from a beam director 98. The light sample comprises lightreflected from the examination substrate 16 and transmitted to the beamdirector 98 by the light transport section 14. As such, the OPS imagingmodule 30 is configured to image the examination substrate 16 usingwither dark field or non-dark filed illumination. Thereafter, the lightsample 40 may encounter a polarizing section 42 having one or moreoptical polarizers therein. The polarizing section 42 permits only lightof a selected or desired polarization to transmit therethrough, therebyfiltering the light reflected by the examination substrate 16 andimproving image quality. IN an alternate embodiment, the OPS imagingmodule 30 may incorporate a variety of other optical devices ormethodologies to optimize image quality. The polarized light 44 is thenincident upon a filtering section 46 having one or more optical filterstherein. For example, in one embodiment the filtering section 46contains at least one narrow band pass filter therein configured topermit light within a desired wavelength range to be transmittedtherethrough. Exemplary narrow band pass filters include, withoutlimitation, from about 380 nm to about 450 nm (violet filter), fromabout 445 nm to about 510 nm (blue filter), from about 495 nm to about580 nm (green filter), from about 575 nm to about 595 nm (yellowfilter), from about 590 nm to about 625 nm (orange filter), from about615 nm to about 710 nm (red filter), and from about 690 nm to about 910nm (color or photo infrared filter). Optionally, the OPS imaging section30 may include filters enabling ultraviolet radiation to transmittherethrough. In an alternate embodiment, the filtering section 46receives light from the light transport section 14 prior to the lightsample 40 being polarized.

Referring again to FIG. 2, the filtered light 48 is then transmittedfrom the filtering section 46 to an image capture device 50. Exemplaryimage capture devices 46 include, without limitations, charge coupleddevices (CCD) and photomultiplier devices. For example, in oneembodiment a CCD chip having about 1000 by 1000 pixel resolution orhigher may be used. Optionally, images captured at various wavelengthsmay be captured and compared to permit image normalization. In analternate embodiment, an image capture device 50 may be utilized tocorrect for motion effects and aberrations. The image capture device 50forms an image of light reflected from the examination substrate 16 andtransmitted to the OPS imaging section 30 by the light transport section14. (See FIG. 1). In the illustrated embodiment, the image capturedevice 46 is in communication with a processor and display device 52.The processor and display device 52 may be used to process informationfrom the image capture device 50 and display the information in anynumber of ways. Exemplary processor and display devices include, withoutlimitations, computers and display terminals.

As shown in FIG. 2, the OPS section 30 may include a light modulator 54and/or an OPS optics suite 56. The light modulator 54 may be used tosegment the sample light 40, thereby providing a stroboscopic effectthereto. Exemplary light modulators 54 include, without limitations,light choppers, shutters, and light valves including liquid crystallight valves. An OPS optics suite 56 may be used to focus, defocus,collimate, or otherwise refine the light sample 40 transmitting throughthe OPS imaging section 30. Exemplary components which may be usedwithin the OPS optics suite 56 include, without limitations, mirrors,positive lenses, negative lenses, acromats, compound lenses, astigmats,windows, flats, adaptive optics, holographical optical elements, spatialfilters, pinholes, collimators, stages, and beam splitters. The lightmodulator 54 and the OPS optics suite 56 may be positioned at variouslocations within the OPS imaging section 30.

Referring again to FIG. 2, the reflectance spectrophotometry module 32includes a spectrophotometer 70 coupled to a RFS image processor 72 forcomputing and displaying spectral characteristics of the light reflectedfrom the examination substrate 16. (See FIG. 1). For example, fullspectrum (e.g. white) light is used to illuminate an examinationsubstrate. Thereafter, the light reflected by the examination substrate16 may be captured and the spectral characteristics thereof may beexamined to measure a variety of characteristics of the examinationsubstrate 16, including, without limitation, hemoglobin saturation andhematocrit concentration. Exemplary RFS image processors 72 include,without limitation, CCD and CMOS chips and photo-multiplier devicescoupled to processors and display monitors. As such, thespectrophotometer 70 is in optical communication with the lighttransport section 14. In one embodiment, an RFS optics suite 74 may beused to process and refine the light received from the light transportsection 14. Exemplary components which may be used within the RFS opticssuite 74 include, without limitations, mirrors, positive lenses,negative lenses, acromats, compound lenses, astigmats, windows, flats,adaptive optics, holographical optical elements, spatial filters,pinholes, collimators, stages, wavelength filters, emission filters, andbeam splitters.

As shown in FIG. 2, the fluorescence imaging module 34 includes afluorescence imaging system 90 and a fluorescence image capture device92. Exemplary fluorescence imaging systems 90 may include variety ofoptical components including, without limitation, microscopes, filterwheels, shutters, and optical filters. For example, green, yellow, andclear optical filters may be included. In one embodiment, thefluorescence imaging system 90 is configured to detect fluorescence fromultraviolet (UV) to infrared (IR) wavelengths. The fluorescence imagecapture device 92 may include a variety of devices including, withoutlimitation, CCD chips and photomultiplier devices. Optionally, thefluorescence imaging module 34 may include a fluorescence optical suite94 to refine or otherwise alter the light entering the fluorescencemodule 34. Exemplary components which may be used within thefluorescence optical suite 94 include, without limitations, mirrors,positive lenses, negative lenses, acromats, compound lenses, astigmats,windows, flats, adaptive optics, holographical optical elements, spatialfilters, pinholes, collimators, stages, wavelength filters, emissionfilters, and beam splitters.

Referring again to FIG. 2, a beam director 98 may be included within orproximate to the analyzing section 12 and configured to direct lightfrom the light transport section 14 to the OPS imaging module 30, thereflectance spectrophotometry module 32, and/or the fluorescence imagingmodule 34. Exemplary beam directors 98 include, without limitation,mirrors including dichroic mirror or elements and dark field mirrors,beam splitters, optical switches, movable or spinning geometric mirrors,corner cubes, prisms, and optical gratings. For example, in oneembodiment the beam director 98 comprises a beam splitter directingfifty percent of the incoming light to the OPS imaging module 30 and 50percent of the incoming light to the reflectance spectrophotometrymodule 32. In an alternate embodiment, the beam director 98 comprises amirror having a non-reflecting area formed thereon, thereby reflecting aportion of light to the spectrophotometer and permitting dark fieldillumination to the OPS imaging module 30 and/or fluorescence imagingmodule 34. Optionally, the beam director 98 may comprise a spinning ormoving mirrored polygon configured to reflect light from the lighttransport section 14 to the OPS imaging module 30, the reflectancespectrophotometer module 32, and/or the fluorescence imaging module 34.In another embodiment, the beam director 98 may be selectively actuatedby the user to direct light to at least one of the OPS imaging module30, the reflectance spectrophotometer module 32, the fluorescenceimaging module 34, and/or any additional modules 34 coupled to or inoptical communication with the analyzing section 12.

In one embodiment of the imaging system 10, the OPS imaging module 30 iscoupled to the light transport section 14, while the reflectancespectrophotometer module 32 and/or the fluorescence imaging module 34are positioned external to the imaging system 10 in opticalcommunication therewith. A beam director 98 is positioned within the OPSmodule 30 and configured to direct a percentage (e.g. fifty percent) ofthe light received by the analyzing section 12 along an optical path tothe reflectance spectrophotometer module 32 and the fluorescence imagingmodule 34, while the remaining light is directed to the OPS imagingmodule 30. An external beam director (not shown) may be used to furtherdivide the directed light between the reflectance spectrophotometermodule 32 and the fluorescence imaging module 34.

FIGS. 1 and 3 show an embodiment of a light transport section 14 of animaging system 10. In the illustrated embodiment, the light source 20 ispositioned proximate to a first lens 100. A variety of light sources maybe used to illuminate the examination substrate 16, including, withoutlimitation, incandescent lamps, gas discharge lamps, dye lasers, solidstate devices such as light emitting diodes, laser diodes, gas lasers,excimer lasers, solid states lasers, and chemical lasers. For example,in one embodiment the external light source 20 comprises an incandescentlamp configured to irradiate the examination surface 16 with whitelight. In an alternate embodiment, the external light source 20comprises a mercury lamp thereby stimulating fluorescence in the tissueof the examination substrate 16. In still another embodiment, the lightsource 20 comprises one or more LEDs configured to illuminate theexamination substrate 16 with light of a discreet wavelength. In stillanother embodiment, the light transport section 14 may include a numberof light sources. For example, a white light source and a UV lightsource could be used simultaneously. When using multiple light sources ashutter or beam splitter may be used to operate the system with adesired light source. For example, to operate the system using the whitelight source a shutter could be positioned to prevent the UV light fromentering the light transport section 14. Thereafter, the user mayactuate the shutter to illuminate the examination substrate 16 with theUV light rather than white light. In an alternate embodiment, a lasersource may be coupled to or in optical communication with the imagingsystem 10 to treat the examination substrate 16. For example, the lasersource may be used to treat microcirculatory disorders including,without limitation, cancerous tissue, skin discolorations, and/or tissuelesions. Optionally, the imaging system 10 may be operated without afirst lens 100.

Referring again to FIGS. 1 and 3, light emitted by the external lightsource 20 is incident on a polarizer 102 configured to polarize light toa desired orientation. Thereafter, polarized light rays 104A, 104B, and104C are incident on a light director 106 configured to direct lightrays 104A′, and 104B′ to the examination substrate 16. In theillustrated embodiment, the light director 106 includes a non-reflectiveor dark field spot 108 formed thereon, thereby permitting light ray 104Cto proceed therethrough and be absorbed by a beam dump or absorber 110.Exemplary light directors include, without limitation, beam splitters,dichroic junctions, and mirrors.

A light guide 112 in optical communication with the light source 20receives and transmits light rays 104′A, 104B′ to the examinationsubstrate 16. In the embodiment illustrated in FIG. 3, the light guide112 includes an illumination segment 114 and a reflectance segment 116.The illumination segment 114 transmits light to the examinationsubstrate 16 for illumination, while the reflectance segment 116transmits reflected light from the examination substrate 16 to the beamdirector 98 of the analyzing section 12. Exemplary light guides include,for example, boroscopes, endoscopes, liquid light guides, polymer lightguides, glass light guides, tubular bodies, and single or bundledoptical fibers. For example, FIGS. 4A-4D show several embodiments oflight guides 112 which may be used with in the light transport section14. As shown in FIG. 4A, the light guide 112 may include polymerillumination and reflectance segments 114, 116, respectively. Thereflectance segment 116 may be optically isolated from the illuminationsegment 114, for example, by an internal cladding 115. Similarly, theillumination segment 114 may include an external cladding 117 thereon.As shown in FIG. 4B, the reflectance segment 116 may be comprised of abundle of optical fibers while the illumination segment 114 comprises apolymer light guide. In the alternative, FIG. 4C shows a light guide 112having an illumination segment 114 constructed of a bundle of opticalfibers and having a polymer reflectance segment 116 therein. FIG. 4Dshows another embodiment wherein the illumination segment 114 and thereflectance segment 116 are constructed from a bundle of optical fibers.

As shown in FIG. 3, a lens or lens system 118 may be included within thelight transport section 14 to focus the light rays 104A′, 104B′. Thefocal point 120 of the lens system 118 may be located above, at thesurface of, or below the surface of the examination substrate 16.Optionally, the lens system 118 may include a reflector or other deviceconfigured to project illuminating light at any angle relative to thelongitudinal axis of the light transport body 14. For example, the lenssystem 118 may permit a user to project light at an angle of about 90degrees relative to the longitudinal axis of the light transport body14. As shown, the distal tip 137 of the light transport body 14 ispositioned a distance D from the examination substrate 16. As a result,the light transport body 14 does not contact the examination substrate16 thereby permitting the unimpeded flow of material through theexamination substrate 16. As such, the imaging system 10 permits theuser to measure the flow of a material through the examination substrate16 in real time. Light 122 reflected from the examination substrate 16is captured by the lens 118 and transmitted through the reflectancesegment 116 and the dark field spot 108 of the light director 106 to thebeam director 98 analyzing section 12. Optionally, a polarizer (notshown) may be positioned proximate to the distal tip 137 of the lighttransport body 14 and configured to polarize light prior to illuminatingthe examination substrate 16.

FIG. 5 shows an alternate embodiment of a light transport section 14. Asshown, an internal light source 18 may be used to illuminate theexamination substrate 16. For example, one or more LEDs may be used toilluminate an examination substrate 16 with a discreet wavelength oflight. In an alternate embodiment, the internal light source 18 maycomprises LEDs of different color, thereby illuminating the examinationsubstrate 16 with light of multiple discreet wavelengths or with fullspectrum light for additional treatment (e.g. laser ablation). Multiplewavelength LED's can also be used to generate images of the distributionof Hb saturation in an SDF imaging modality. Those skilled in the artwill appreciate that the use of LEDs as a light source enables theimaging system 10 to be powered by a battery or other low-power powersupply relative to previous systems. For example, the imaging system 10may be powered by coupling the imaging system 10 to a universal serialport of a personal computer. One or more internal lenses 130 may, butneed not be, included within the light transport section 14 andpositioned proximate to the internal light source 18. Similarly, one ormore optical polarizers or filters 132 may be positioned proximate tothe internal lenses 130. The internal light sources 18 emit rays 134A,134B which are transmitted to the examination substrate 16 by theillumination segment 136 of the light guide 138. An examination lenssystem 140 may be used to focus the light rays 134A, 134B to theexamination substrate 16. A focal point 142 of the lens system 140 maybe located above, at the surface of, or below the surface of theexamination substrate 16. Thereafter, light rays 144 reflected by theexamination substrate 16 are collected by the lens system 140 andtransmitted to the beam director 98 of the analyzing section 12 by thereflectance segment 146 formed within the light guide 138.

FIG. 6 shows an alternate embodiment of a light guide 150. As shown, thelight guide 150 includes an illuminating segment 152 having a focused orcurved distal tip 154, thereby directing light rays to a focal pointwithin an examination substrate (not shown). The reflectance segment 156is configured to transmit light from the examination substrate 16 to theanalyzing section 12 (See FIG. 1).

FIGS. 7 and 8 show embodiments of spacer devices which may be affixed tothe distal end or distal section of the light guide. FIG. 7 shows alight guide 160 having a spacer 162 attached thereto. In the illustratedembodiment, the distal section of the light guide 160 may include one ormore lock members 164 thereon to securely couple the spacer 164 to thelight guide 160. As such, the spacer 160 may include a locking memberrecess 166 to accommodate the locking members 164. The spacer 162ensures that the light guide 160 remains at least a distance d from theexamination substrate 16. FIG. 8 shows an alternate embodiment of aspacer 172 coupled to a light guide 170. The spacers 162, 172 may bemanufactured from a variety of materials including, without limitation,plastic, rubber, elastomer, silicon, or any other biologicallycompatible material. In one embodiment, the spacer 162, 172 aredisposable.

FIGS. 9 and 10 show an embodiment of a light guide 180 having analternate embodiment of a spacer 182 attached thereto. The spacer 182includes a vacuum port 184 attachable to a source of vacuum (not shown).The spacer 182 includes a spacer aperture 186 for irradiating theexamination substrate (not shown). The spacer 182 includes one or moreattachment orifices 188 thereon which are in communication with thevacuum port 184. The attachment orifices 188 are formed between anexterior wall 190 and an interior wall 192 of the spacer body 194 andare isolated from the spacer aperture 186. As such, the spacer 180 isconfigured to couple to the examination substrate (not shown) when thevacuum source is actuated without adversely effecting the irradiation ofthe examination surface. As such, the spacer 180 may be rigid or, in thealternative, may be constructed of a compliant material for use withinor on compliant organs or structures. Like the embodiments describedabove, the spacer 182 may be manufactured from a variety of materialsand may be disposable. One or more additional ports may be formed on thespacer body 194 for the administration of medicinal or therapeuticagents.

FIGS. 11 and 12 show an embodiment of an imaging system 200. As shown,the imaging system 200 includes an illumination body 202 and areflectance body 204. The illumination body 202 defines an optics recess206 configured to receive an optical system 208 therein. The opticalsystem 208 includes a first spacer 210, a first dark field mirror 212, afilter spacer 213, and a filter bank 214. In the illustrated embodiment,the filter bank 214 includes a clear filter 216, a yellow filter, 218, agreen filter 220, and a white filter 222. A second spacer 224 ispositioned proximate to the filter bank 214. A third spacer 226 ispositioned between the second spacer 224 and a lens 228. A fourth,fifth, and sixth spacers 230, 232, and 234, respectively, are positionedproximate thereto. A second dark field filter 236 is positioned betweenthe sixth spacer 234 and the seventh spacer 238.

Referring again to FIG. 11, the reflectance body 204 includes a lightdirector 240 therein. The light reflector 240 includes a non-reflectivearea 242 formed thereon. In addition, the reflectance body 204 includesan examination tip 244 which is configured to be positioned proximate tothe examination substrate (not shown). A polarizer and/or filter 246 andan image capture device 248 may be positioned within the analyzingsection 250 of the reflectance body 204. During use, a light source 252projects light which is filtered and focused by the optical system 208located within the illumination body 202. The light from the lightsource 252 is directed by the light director 240 to the examinationsubstrate (not shown) located proximate to the examination tip 244.Light reflected by the examination substrate (not shown) is transmittedto the analyzing section 250 by the light guide 256, where the light isdepolarized and analyzed.

FIG. 13 shows another embodiment of an imaging system. As shown, theimaging system 300 includes an illumination body 302 and a reflectancebody 304. The illumination body 302 defines an optics recess 306configured to receive an optical system 308 therein. The optical system308 includes a first lens 310 and a second lens 312. Positionedproximate to the first lens 310 is an internal light source 318. In theillustrated embodiment, the internal light source 318 comprises a numberof LEDs configured to project light through the optical system 308. Oneor more reflectors 316 may be used to ensure that the light istransmitted through the illumination body 302.

As shown in FIG. 13, the reflectance body 304 includes a light director340 therein. The light reflector 240 includes a non-reflective area 342formed thereon. In addition, the reflectance body 304 includes anexamination tip 344 which is configured to be positioned proximate tothe examination substrate (not shown). A beam director 398 and an imagecapture device 348 may be positioned within the analyzing section 350 ofthe reflectance body 304. During use, the light source 318 projectslight which is focused by the optical system 308 located within theillumination body 302. The light from the light source 318 is directedby the light director 340 to the examination substrate (not shown)located proximate to the examination tip 344. Light reflected by theexamination substrate (not shown) is transmitted to the analyzingsection 350 by the light guide 356, where the light is analyzed. Asshown, the analyzing section 350 may include one or more filters orpolarizers 360 therein.

As shown in FIGS. 3-5, at least one light source may be used toilluminate structures located below the surface of a substrate. FIGS. 14and 15 show alternate embodiments of imaging systems useful in imagingsub-surface structures while avoiding or reducing the effects of surfacereflection. FIG. 14 shows an imaging system 400 comprising a body 402having one or more imaging passages 404 formed therein. One or moreillumination passages 406 may be formed within the body 402 and may beoptically isolated from the imaging passage 404. In one embodiment, thebody is rigid. In an alternate embodiment, the body 402 is flexible. Forexample, the body 402 may comprise a catheter body. Optionally, the body402 may include an additional lumen formed therein. For example, anadditional lumen may be positioned within the body 402 and may be usedto deliver therapeutic agents to a treatment site. In anotherembodiment, an additional lumen may be used to deliver a vacuum force toa treatment site. In the illustrated embodiment, the illuminationpassage 406 is positioned radially about imaging passage 404. In theillustrated embodiment, the illumination passage 406 encircles theimaging passage 404. In an alternate embodiment, the illuminationpassage 406 may be positioned anywhere within the body 402. As shown,the illumination passage 406 is optically isolated from the imagingpassage 404. Therefore, illuminating energy transported through theillumination passage 406 is prevented from entering the imaging passage404. As such, the present systems permits side stream dark field imaging(hereinafter SDF). As shown in FIG. 14, a feature of SDF imaging is thatthe illuminated light 412A and 412B and the reflected light 414 travelvia independent pathways. Thus, the illumination can be placed directlyon the tissue and the observations can be made adjacent to it withoutlight crossing over between two paths.

Referring again to FIG. 14, at least one illumination source may bepositioned within the illumination passage 406. In one embodiment, theillumination source 410 comprises one or more LED's configured toproject a selected wavelength to the substrate 420. In an alternateembodiment, the illumination source 410 comprises a plurality of LED'sconfigured to project multiple wavelengths to the substrate 420. Forexample, as shown in FIG. 14 a first illumination source 410A configuredto project light to the substrate 420 is positioned at the distalportion 418 of the body 402. Similarly, a second illumination source410B is positioned at the distal portion 418 of the body 402. As such,the first and second illumination sources 410A, 410B are positionedproximate to the substrate 420 under examination. Optionally, any numberof illumination sources may be positioned within the body 402. Exemplaryillumination sources include, without limitation, LED's, LLED's,incandescent bulbs, laser light sources, etc.

FIG. 15 shows a perspective view of the distal portion of an alternateembodiment of the imaging device 400 shown in FIG. 14. As shown, thebody 402 includes an imaging passage 404 and at least one illuminationpassage 406 optically isolated from the imaging passage 404. One or moreillumination devices 410 are located within the illumination passage 406and positioned proximate to the distal portion 418 of the body 402. Assuch, during use the illumination source are positioned proximate to thesubstrate 420.

FIG. 16 shows a cross sectional view of the distal portion of anembodiment of an imaging device. As shown, The body 402 defines animaging passage 404 and an illumination passage 406 therein. Like theprevious embodiments, the illumination passage 406 is optically isolatedfrom the imaging passage 404. In the illustrated embodiment, theillumination passage 406 terminates proximate to the distal portion 418of the body 402. Optionally, the illumination passage 406 may continuethrough the length of the body 402. As such, the illumination passage406 may include one or more optical fibers configured to deliverilluminating energy to the substrate 420 from a remote location. In theillustrated embodiment, one or more illumination sources 410 arepositioned within the illumination passage 406. For example, one or moreLED's may be positioned within the illumination passage 406. Like theprevious embodiments shown in FIGS. 14 and 15, the illumination passage406 is optically isolated from the imaging passage 404. Optionally, atleast one conduit 424 may traverse through the body 402 thereby couplingthe illumination source 410 to a source of power. In the illustratedembodiment at least one lens 422 is positioned within the imagingpassage 404 thereby transmitting an image received from a substrate 420to an image capture device 416. (See FIG. 14). Optionally, the imagingsystem shown in FIGS. 14-16 may be used without a lens 422.

With reference to FIG. 14, during use, the first illumination source410A projects illuminating energy 412A to the substrate 420. Similarly,the second illumination source 410B projects illuminating energy 412B tothe substrate 420. As shown in FIG. 14, the illumination energies 412A,412B are optically isolated from the imaging passage 404. The first andsecond illumination energies 412A, 412B may be the same or differingwavelengths. Further, as the first and second illumination sources 410A,410B are positioned at the distal portion of the body 402 proximate tothe substrate 420, surface reflections therefrom are reduced oreliminated. As shown, a sub-surface image 414 is transported by theimaging passage 404 from the substrate 420 to an image capture device416. Exemplary image capture devices include, without limitation, CCDdevices, cameras, spectrophotometers, photomultiplier devices,analyzers, computers, etc. Optionally, one or more lenses 422 may bepositioned within the image passage 404 or body 402 to focusillumination energy 412 to the substrate 420 or to assist in thetransport of an image 414 from the substrate 420 to the image capturedevice 420416, or both. As stated above, the optical isolation of theillumination energy from the image received from the substrate reducesor eliminates the effects of surface reflections while enabling SDFimaging in addition to a variety of alternate imaging modalities orspectroscopic examination of an area.

FIG. 17 shows an alternate embodiment of an SDF imaging system. Asshown, the SDF imaging system 450 comprises a body 452 defining animaging passage 454 and an illumination passage 456 optically isolatedfrom the imaging passage 454. The illumination passage 456 includes oneor more illumination sources 460 therein. Exemplary illumination sources460 include, without limitation, LED's, LLEDs, and incandescent bulbs.As shown, the illumination sources 460 are located proximate to thedistal portion 462 of the body 452. Optionally, the illumination sources460 may be located some distance from the examination area. As such,illuminating energy may be transported to the examination area throughfiber optic conduits positioned within the body 452. Like the previousembodiments, the body 402 may be rigid or flexible. In the illustratedembodiment, a cap device 464 is positioned over the body 452. In oneembodiment, the cap device 464 may comprise an optically transparentdisposable cap device 464 configured to be detachably coupled to thebody 452. During use the cap device 464 may protect the body 402 frombiological materials and contaminants. As such, the cap device 464 maybe sterile.

Referring again to FIG. 17, at least one lens 466 may be positionedwithin the imaging passage 454. The imaging passage 454 is in opticalcommunication with an imaging capture device 468. The image capturedevice 468 may comprise any of devices useful in capturing and analyzingan image received from a substrate. For example, the image capturedevice 468 may comprise a CCD device, photomultiplier, computer,spectrophotometer, and the like. Further, a focusing device 470 may beincluded within the body 452 or the image capture device 468. Exemplaryfocusing devices include, without limitation, additional lenses,mechanical drives or positioners, and the like. Optionally, the SDFimaging system 450 may further include a handle 472 to assist a user inpositioning the device. Further, the SDF imaging system 450 may beconfigured to be coupled to a computer, power source, etc.

FIG. 18 shows an alternate embodiment of an imaging system. As shown,the imaging system 500 includes a body 502 defining an imaging passage504 and at least one illumination passage 506 optically isolated fromthe imaging passage 504. The illumination passage 506 includes one ormore illumination sources 510 therein. As shown, the illuminationsources 510 are located proximate to the distal portion 518 of the body502, however, the illumination source may be located anywhere on thebody 502. Optionally, a cap device (not shown) may be positioned overthe body 502. For example, the cap device (not shown) may comprise anoptically transparent disposable device configured to be detachablycoupled to the body 502.

Referring again to FIG. 18, at least one lens 522 may be positionedwithin the imaging passage 504. The imaging passage 504 is in opticalcommunication with at least one image capture device 516. In theillustrated embodiment, a first image capture device 516A and a secondimage capture device 516B may be used with the system. Further, one ormore optical modulators 526 may be positioned within the image passage504 and configured to modulate imaging signals from the substrate 520.Exemplary optical modulators 526 include, without limitation, mirrors,band pass plates, polarizers, gratings, and the like. The image capturedevices 516A, 516B may comprise any number of devices useful incapturing and analyzing an image received from a substrate. For example,the image capture devices 516A, 516B may comprise CCD devices,spectrophotometers, spectrum analyzers, and the like.

As shown in the FIGS. 18 and 19, the illumination sources 510 maycomprise LED's of a single wavelength. In the alternative, theillumination sources 510 may be configured to irradiate light ofmultiple wavelengths. For example, FIG. 19 shows a device having a firstillumination source 510A irradiating at a first wavelength and a secondillumination source 510B irradiating at a second wavelength. FIG. 20shows a device having a first illumination source 510A, a secondillumination source 510B, and a third illumination source 510C, eachillumination source irradiating at a different wavelength. As such, thesystem may be configured to perform a number of imaging and analyzingprocedures with a single device. For example, a first wavelength may beprojected to the substrate and used for SDF microcirculation imagingwithin the underlying vasculature, while a second wavelength may beprojected to the substrate and used for detecting oxygen saturationwithin a blood flow. In short any number of wavelengths of illuminatingenergy may be projected from the illumination sources 510 and used forany number of analytical processes. For example, the imaging system 500may be configured to permit imaging of the microcirculation andspectroscopic examination of an area with a single device.

Referring to FIGS. 18 and 21, during use the distal portion 518 of thebody 502 may be in contact with the substrate 520 under examination. Assuch, the illumination source(s) 510 may be positioned in closeproximity to the substrate 520. Optionally, the distal portion 518 mayinclude one or more engaging devices 528 coupled to the body 504 or thecap device (not shown). For example, as shown in FIG. 22, the engagingdevice 528 may comprise an inflatable device configured to dissipate apressure applied to the substrate 520 by the distal portion 518 of thebody 502. In an alternate embodiment, shown in FIG. 23, the distalportion 518 may be positioned proximate to, but not in contact with, thesubstrate 520. As such, the illumination sources 510 may be configuredto project illuminating energy 512A, 512B to the substrate 520.Optionally, one or more lenses may be in optical communication with theillumination source(s) 510 to aid in the projection of illuminationenergy 512A, 512B to the substrate 520.

FIG. 24 shows a block diagram of an embodiment of an imaging andanalyzing system. As shown, the imaging system 600 includes an analyzingsection 602, a light transport section 604, and a light delivery section606 configured to deliver light to and receive information from asubstrate 608. As stated above, the analyzing section 602 may includeany number of analyzing modules configured to process informationreceived from the substrate 608. In the illustrated embodiment, theanalyzing section 602 includes an OPS imaging module 620, a dark filedillumination module 622, a reflectance spectrophotometry module 624, anadditional processor module 626, a fluorescence module 628, and/or afluorescence lifetime module 630. The additional processor module 626may include one more processing module including, without limitation,Raman spectroscopy devices, fluorescence decay processors, PpIXanalyzers, and/or OCT (Hb sat) analyzers, and/or CO2 analyzers.Referring to FIGS. 18 and 21, the analyzing section 602 may beconfigured to receive imaging information from the substrate 520 via theimaging passage 504 formed within the body 502. Those skilled in the artwill appreciate that the present system enables a user to selectivelyanalyze a substrate using multiple imaging modalities, spectrophotometrymodalities, and similar analyzing methods using a single device coupledto multiple analyzers.

The light transport section 604 may comprise a body 634 configured totransport light to and from the substrate 608. For example, the body 634may include an image passage 504 and an optically isolated illuminationpassage 506 as shown in FIG. 18. Further, the light transport section604 may include one or more internal illumination sources 636 positionedtherein and configured to irradiate the substrate 608. Optionally, oneor more optical elements 634 may be positioned within the body 632.Further, the body 632 may be configured to receive and transport lightfrom an external light source 638 to the substrate 608.

Referring again to FIG. 24, the light delivery section 606 may comprisea direct illumination source 640 configured to be positioned proximateto the substrate 608 and providing direct illumination thereto. As such,the direct illumination source 640 is optically isolated from an imagereceived from the substrate 608. Exemplary direct illumination sources640 include LLED's, LED's, and the like. Further, one or more whitelight illumination sources 642 may be used to illuminate the substrate608. In one embodiment, the light delivery section 606 may be configuredto deliver materials to or receive materials 644 from the substrate 608.For example, the light delivery section 606 may be configured to infusetherapeutic agents to the substrate 608. Optionally, the light deliverysection 606 may include one or more engaging devices 646 positionedthereon to assist in positioning the system during use.

As stated above, the preceding imaging and analyzing systems disclosedherein may include one or more cap devices 464 which may be detachablycoupled to the body 452. (See FIG. 17). Generally, the cap device 464may comprise optically transparent materials configured to protect thebody 452 during use. As such, the cap device 464 may be disposable. FIG.25 shows an alternate embodiment of a cap device 714. As shown in FIG.25, the imaging device 700 includes a body 702 having an imaging passage704 and an illumination passage 706 optically isolated from the imagingpassage 704 formed therein. The illumination passage 706 may include oneor more conduits 708 coupled to one or more illumination sources 710located therein. As shown in FIG. 25, one or more lenses 712 may bepositioned within the imaging passage 704. A cap device 714 may becoupled to the body 702. The cap device 714 includes an illuminationfield 716 optically isolated from an imaging relief 718. In theillustrated embodiment, the illumination field 716 is positionedproximate to the illumination sources 710 located within the body 702.Similarly, the imaging relief 718 is positioned proximate to the imagingpassage 704. At least one isolation surface 720 optically isolates theillumination field 716 from the imaging relief 718. For example, in theillustrated embodiment the isolation surface 720 include a reflectivefoil 722 thereon which is configured to prevent light from illuminationsources 710 from directly entering the imaging passage 704 without firstengaging a substrate under examination. Alternate isolation materialsmay be used on the isolation surface 722 including, without limitation,dyes, foils, impregnations, etc. Optionally, the cap device 714 may bedisposable and may be configured to detachably couple to the body 702.

FIG. 26 shows yet another embodiment of a reflectance avoidance imagingsystem. As shown, the reflectance avoidance imaging system 810 includesan imaging device 812 having a spacer or tissue engaging tip 814attached thereto. The imaging device 812 includes a body 816 having adistal portion 818 configured to receive and engage the spacer 814. Inone embodiment, the spacer 814 is detachably coupled to the body 816.Optionally, the spacer 814 may be non-detachably coupled to the body816.

Referring again to FIG. 26, the imaging body 816 includes one or moreconduits formed therein. In the illustrated embodiment, the body 816includes an imaging conduit 820 configured to project light from a lightsource (not shown) to a work surface. In addition, the imaging conduit820 collects light reflected from the work surface and transports thereflected light to a sensor suite (not shown) in communicationtherewith. Exemplary sensor suites include, without limitation, a CCD orany other type of imaging or sensing device, spectral photometers, andthe like. Optionally, a secondary imaging conduit 822 may be positionedwithin the body 816. For example, the secondary imaging conduit 822 maybe configured to measure CO₂ within tissue through the use of a CO₂sensing dye. The CO₂ sensing dye enables the measurement of fluorescencedecay and may utilize light received from and transmitted through theimaging conduit 820. Optionally, one or more additional conduits 824 maybe positioned within the body 816. For example, any number of fluidconduits may be formed within the body 816.

The spacer 814 includes a spacer body 830 having a coupling portion 832configured to engage and couple the distal portion 818 of the body 816.The spacer body 830 further defines an orifice 834 which is incommunication with the coupling portion 832. In the illustratedembodiment, the spacer body 830 includes thread members 836 andattachment devices 838 formed or otherwise disposed thereon to enablethe spacer body 830 to couple to the body 816. Any number or type ofthread members 836 and attachment devices 838 may be used to couple thespacer body 830 to the imaging device 812. The distal portion of thespacer body 830 includes a flange 840 defining the orifice 834. In theillustrated embodiment, the flange 840 includes one or more vacuum ports842 portioned thereon, thereby permitting the flange 840 to engage orcouple to the a work surface.

In the illustrated embodiment, the spacer 814 includes one or morevacuum ports 842 which enable the spacer 184 to engage the work surface.Optionally, the spacer body 814 may be configured to avoid contactingthe work surface. For example, the spacer body 814 may include anoptical system comprised of one or more lenses to enable the imagingdevice 812 to project and receive light to and from a work surface froma distance without contacting the work surface. For example the opticalsystem may include a zoom lens system.

Further, the spacer 814 may be formed in any variety of shapes and size.For example, the spacer may include a doughnut-shaped spaces.Furthermore, the spacer 814 may include a bladder or cushion filled withany variety of fluids. Optionally, the fluid may be opticallytransparent.

FIG. 27 shows a cross sectional view of an embodiment of a reflectanceavoidance imaging system 810. As shown, the body 816 includes theimaging conduit 820, the secondary imaging conduit 822, and the additionconduit 824 formed therein. In addition, vacuum conduits 856 and 858 areformed within the body 816 and a couple to a vacuum source. (not shown)The spacer 814 includes vacuum conduits 870 and 872 which are incommunication with the vacuum conduits 856 and 858 of the body 816 andthe vacuum ports 842 formed on the spacer 814. Optionally, one or moreattachment members 862 may be positioned on the body 816 to furtherenable coupling of the spacer 814 to the body 816.

In addition to the novel imaging devices described above, the presentapplication describes a method of imaging and determining variousbiological parameters non-invasively and, if needed, treating anaffected area. For example, when operating the above-described system inan OPS imaging mode, flow though the capillaries and related circulatorystructures may be examined be viewing red blood flow therethrough. Tooperate the system in an OPS imaging mode, the user irradiates theexamination substrate with white light. The white light is polarized bya polarizer prior to illuminating the examination substrate. Reflectedlight is captured by the light guide and transmitted to the polarizingsection 42 of the OPS imaging module 30 (See FIG. 2). Light reflected bythe system optics and the patient's tissue surface undergoes apolarization shift as a function of scattering and, thus, is cancelledby the polarizing section 42. As such, sub-surface reflected light failsto undergo a polarization shift and will be captured by the imagecapture device 50, thereby enabling sub-surface imaging. Optionally, OPSimaging may be accomplished in combination with dark field illumination.

Similarly, the imaging system described herein may be used to performreflectance spectrophotometry using the reflectance spectrophotometrymodule. A spectrophotometer may be used with the present imaging systemto examine the spectral reflectance of the tissue surface. Light from alight source illuminates an examination substrate. The light maycomprise an internal light source 18, external light source 20, and/oran ancillary light source 22. (See FIG. 1). Light reflected by theexamination substrate 16 is captured by a light transport section 14 andtransmitted to a reflectance spectrophotometry module. The spectralcharacteristic of the reflected light may then be examined and used todetermine the hemoglobin saturation, and/or hematocrit concentrationwithin the surface of an organ under investigation.

Lastly, the imaging system described herein may be used to determine theoxygenation and/or functional state of a tissue cell using thefluorescence imaging module. For example, an examination area may beilluminated with UV light thereby targeting the mitochondrial energystate therein. For example, light having a wavelength of about 360 nmmay be used to illuminate the examination substrate. Thereafter, lightreflected by the substrate may be captured by the light transportsection 14 and transmitted to the analyzing section 12. (See FIG. 1) Thecaptured light may undergo a lambda shift from 360 nm to about 460 nm.Thereafter, a fluorescence imaging module 34 may analyze the reflectedlight for to determine the presence of NADH in the cells, therebyshowing availability of oxygen within the cells.

The OPS imaging processor 52, RFS processor 72, and fluorescence imagingprocessor 92 may each contain any number of formulas, algorithms,models, databases, look-up tables, or related information to compute anddisplay their respective reflectance measurements. For example,Beers-Lambert law may be used to determine the concentration of materialin the examination substrate based on the absorbance of the light by theexamination substrate.

Also disclosed herein is a method of comprehensively monitoring themicrocirculation of a patient. The method may include using any of theaforementioned imaging systems disclosed herein. In one embodiment, themethod includes illuminating a tissue substrate, avoiding the reflectionof light from the surface of the tissue substrate, receiving light fromthe tissue substrate, utilizing some of the received light to imagemicrocirculatory flow in the tissue substrate, utilizing some of thereceived light to determine oxygen availability in the microcirculation,and utilizing some of the received light to determine the adequacy ofoxygenation of the tissue cells.

In one embodiment, the aforementioned method may include utilizing themicrocirculatory flow information, the oxygen availability information,and the adequacy of oxygenation of tissue cells information, making anearly and sensitive determination regarding states of shock, such asseptic, hypovolemic, cardiogenic and obstructive septic shock, inpatients, and guiding resuscitation therapies aimed at correcting thiscondition.

In another embodiment the aforementioned method may also includeutilizing the microcirculatory flow information, the oxygen availabilityinformation, and the adequacy of oxygenation of tissue cellsinformation, and making an early and sensitive determination regardingcardiovascular disease and failure of the patient.

In closing, it is understood that the embodiments of the inventiondisclosed herein are illustrative of the principals of the invention.Other modifications may be employed which are within the scope of thepresent invention. Accordingly, the present invention is not limited tothat precisely as shown and described in the present disclosure.

What is claimed is:
 1. A handheld system for sidestream dark fieldanalysis of tissue beneath a tissue surface, comprising: an elongatebody portion including an imaging passage extending along a length ofthe elongate body portion; one or more illumination passages which aredisposed radially about the imaging passage within the elongate bodyportion and which are optically isolated from the imaging passage alongan entire length of the imaging passage for surface reflection avoidanceor reduction when an examination tip of the elongate body portion isproximate the tissue surface; a plurality of light sources which aredisposed in optical communication with the one or more illuminationpassages and which are configured to project light onto a first tissuesite via the one or more illumination passages; and an analysis sectionoptically coupled to the imaging passage and configured to capture dataof tissue beneath the tissue surface from a second tissue site differentfrom and adjacent to the first tissue site.
 2. The system of claim 1further comprising at least one isolation surface which is configured tooptically isolate an illumination field of the first tissue site from animaging field of the second tissue site.
 3. The system of claim 1further comprising a disposable detachable cap disposed over a distalportion of the elongate body portion having a distal window configuredto allow illumination and imaging therethrough.
 4. The system of claim 1further comprising an annular spacer disposed on the distal end of theelongate body portion configured to minimize mechanical contact by thedistal portion of the body portion with the second tissue site.
 5. Thesystem of claim 1 wherein the light sources comprise light emittingdiodes.
 6. The system of claim 5 wherein the light emitting diodescomprise green light emitting diodes.
 7. The system of claim 1 furthercomprising a handle secured to the elongate body portion and configuredto be held by a user.
 8. The system of claim 1 wherein the analysissection comprises an image capture device.
 9. The system of claim 8wherein the image capture device comprises a camera.
 10. The system ofclaim 1 wherein the analysis section comprises a spectrophotometrymodule.
 11. The system of claim 1 wherein the analysis section comprisesa fluorescence imaging module.
 12. A method of analyzing tissue in apatient, comprising: providing a system for side stream dark fieldimaging of tissue beneath a tissue surface, including: an elongate bodyportion including an imaging passage extending along a length of theelongate body portion, one or more illumination passages which aredisposed radially about the imaging passage within the elongate bodyportion and which are optically isolated from the imaging passage alongan entire length of the imaging passage for surface reflection avoidanceor reduction when an examination tip of the elongate body portion isproximate the tissue surface, one or more light sources which aredisposed in optical communication with the one or more illuminationpassages and which are configured to project light onto a first tissuesite via the one or more illumination passages, and an analysis sectionoptically coupled to the imaging passage; emitting light from the one ormore light sources onto the first tissue site; gathering light from asecond tissue site through the imaging passage with the analysis sectionand imaging tissue beneath a surface of the second tissue site; andanalyzing light gathered from the second tissue site through the imagingpassage and determining a clinical parameter of the patient.
 13. Themethod of claim 12 wherein analyzing light gathered from the secondtissue site comprises fluorescence imaging.
 14. The method of claim 13wherein analyzing light gathered from the second tissue site comprisesannexing fluorescence imaging.
 15. The method of claim 12 whereinemitting light from the one or more light sources comprises emittinglight from one or more light emitting diodes.
 16. The method of claim 12wherein emitting light from the one or more light sources comprisesemitting light from one or more green light emitting diodes.
 17. Themethod of claim 12 wherein gathering light from a second tissue sitethrough the imaging passage with the analysis section comprisesgathering light from sublingual tissue.
 18. The method of claim 12wherein gathering light from a second tissue site through the imagingpassage with the analysis section comprises gathering light frommicrocapillaries.
 19. A method of analyzing tissue in a patient,comprising: emitting light from one or more light sources of a systemfor side stream dark field imaging through one or more illuminationpassages of the system which are disposed radially about an imagingpassage within an elongate body portion of the system and which areoptically isolated from the imaging passage along an entire length ofthe imaging passage and onto a first tissue site; gathering light from asecond tissue site through the imaging passage with an analysis sectionof the system and imaging tissue beneath a surface of the second tissuesite; and analyzing light gathered from the second tissue site throughthe imaging passage and determining a clinical parameter of the patient.